PROJECT SUMMARY/ABSTRACT A growing body of evidence indicates that molecular signaling mechanisms mediated by the myddosome complex in kidney stromal cells, drive fibrosis triggered by interleukin 1b (IL1b) and Toll-like receptor (TLR) ligands. Work from our laboratory and by others has shown that signaling mechanisms associated with IRAK4, a key component of the stromal cell myddosome, contributes to the development of renal fibrosis after acute kidney injury. IRAK4, therefore, emerges as a suitable target for much needed therapies targeting renal fibrosis. In order to effectively target IRAK4, however a deeper understanding of its mechanisms is needed. IRAK4 has been shown to possess two distinct functions, one as a serine/threonine kinase and the other as a structural scaffold necessary for myddosome formation. Importantly, the role of IRAK4 as a myddosome scaffold is necessary for its kinase activity, but the latter is not necessary for myddosome-mediated signaling. We have reported that pharmacologic inhibition of IRAK4 kinase activity with a selective small molecule significantly reduces pro-fibrotic stromal cell activity, including proliferation and differentiation into myofibroblasts, both ex vivo in response to IL1? stimulation, and in vivo after ischemic kidney injury. Our data further indicated that those profibrotic mechanisms depend on stabilization and activation of the transcriptional regulator MYC via a mechanism involving IL1R-driven autophagy. On the contrary, inhibition of IRAK4 kinase activity did not result in abrogation of synthesis and secretion of NF-?B-regulated inflammatory cytokines IL1? and IL6 in kidney stromal cells. Those results are in keeping with previous reports indicating that NF-kB activation by IRAK4 is myddosome-mediated and only partially dependent on IRAK4 kinase activity. The objective of this project is to dissect the molecular mechanisms through which IRAK4 mediates kidney fibrosis, by assessing the distinct contribution of kinase-dependent versus myddosome-dependent IRAK4 signaling mechanisms. The long-term goal of these studies is to set the pre-clinical basis for novel therapeutics that can ameliorate both renal fibrosis and local inflammation through blockage of stromal cell pro-fibrotic and pro- inflammatory activities. The central hypothesis is that following acute kidney injury IRAK4 kinase activity in stromal cells is necessary for pro-fibrotic mechanisms, while kinase-independent IRAK4-mediated myddosome assembly is necessary for inflammatory cytokine production by those cells during the process of kidney scarring. Our rationale for the research strategy proposed is based on a combination of genetic ablation strategies to study IRAK4 kinase domain-independent mechanisms, along with cutting-edge bioengineered human kidney organoids for the study of the nephron interstitial microenvironment in the absence of confusing hematopoietic immune signals. In addition, we propose the use of novel therapeutic chemistry shown to be effective for inducible targeted degradation of IRAK4 in vivo, abrogating myddosome formation. Our three specific aims will test three major hypotheses: (Aim 1) IRAK4 kinase function is necessary for profibrotic mechanisms of kidney stromal cells post-AKI; (Aim 2) signaling via IL1R/IRAK4 stabilizes MYC via a mechanism involving stromal cell autophagy and degradation of SQSTM1/P62; (Aim 3) pharmacologic abrogation of IRAK4 using a synthetic protein degrader molecule will impair stromal cell myddosome formation and ameliorate IRI- induced kidney fibrosis. Collectively, these studies will provide critical experimental and mechanistic basis for a rational design of therapies targeting IRAK4 in renal fibrosis.